The invention relates to a chimeric protein wherein HI-8 which is the C-terminal domain of human urinary trypsin inhibitor (UTI) having a cancer cell metastasis inhibitory effect is linked to a peptide containing the G domain of urokinase binding specifically to an urokinase receptor expressed in large amounts on cancer cells.
In current cancer therapy, although advances in early diagnosis and therapy increase a therapeutic rate, an effective remedy against cancer metastasis has not been found. Inhibition of metastasis of cancer is a serious problem. Recent active research clarifies a molecular biological mechanism on metastasis of cancer cells. It has been found that invasion of cancer cells into normal tissue requires actions of a variety of proteases (1) (2). Urokinase-type plasminogen activator (uPA), which is one of serine proteases, is noted earlier as a protease increased with canceration of cells (3). It is reported that the amount of uPA extracted from cancer tissue is generally correlated with malignancy of cancer cells (4). In addition, it is believed that secretion of precursor-type enzymes such as uPA and metalloproteases including collagenase and stromelysin, and a proteolysis cascade including an activation process of the precursor-type enzymes are closely related to an invasion process of cancer cells (5). uPA, which is a glycoprotein having a molecular weight of 55 kDa, has a three-domain structure of, from N-terminal, growth factor-like domain (G domain), kringle domain (K domain) and protease domain (P domain) (see, FIG. 1). G domain is a site to be bound to an urokinase receptor (uPAR) which is a specific receptor on cells (6). It is believed that uPA binds to membrane of cancer cells through the domain and plays an important role during invasion (7) (8) (9) (10) (11). Cancer cells also increase a uPA concentration in the direction to be migrated by collecting uPAR capable of binding to uPA on the tip of migration direction (12). The uPAs bound to cell membrane activate a variety of proteases such as plasminogen on the surface of membrane and degrade extracellular matrices (13) (14) (15).
It is known that plasmin activated by uPA on the surface of membrane of endothelial cell activates latent TGF-xcex2 (transforming growth factor xcex2) which exists on the surface of mural cell (16). It is known that TGF-xcex2 induces production of plasminogen activator inhibitor 1 (PAI-1) which is a selective inhibitory factor of uPA and stimulates expression of mRNA of uPA (17). TGF-xcex2 controls vascularization according to concentration thereof differently.
In view of foregoing, experiments to inhibit metastasis of cancer cell by inhibiting actions of uPA on the membrane of cancer cell have been tried. Reported are inhibition on invasion by antibody (18) or inhibitor (19) against uPA, or, inhibition on invasion by antibody (21) and peptides (22) (23) which inhibit bonding of uPA to uPAR.
An amino terminal fragment (ATF) of uPA (residues 1-135 of uPA) is a polypeptide comprising G domain to be bound to uPAR and adjacent K domain, and competitively inhibits binding of uPA to uPAR. It is reported by Crowley et al. that a chimeric protein comprising a polypeptide containing 137 amino acids from N-terminal including ATF bound to a Fc region of immunoglobulin G is produced and that the protein inhibits metastasis of human cancer cells in vivo (24). Lu et al. prepare a chimeric protein wherein ATF is bound to human serum albumin (HSA) through a spacer consisting of 4 glycines in yeast. They reported that the chimeric protein bound to uPAR in vitro and inhibited binding of uPA to cancer cell membrane (25). These chimeric proteins were produced to stabilize characteristics of ATF having uPA binding inhibitory action in vivo and to increase metastasis inhibitory effects.
Ballance et al. reports a method for producing chimeric proteins in yeast wherein G domain of uPA is bound to plasminogen activator inhibitor-2 (PAI-2) which is an inhibitor of uPA, or, to xcex11-antitrypsin (xcex11-AT) which is a plasmin inhibitor (26). The chimeric protein was produced to increase inhibitory properties by combining G domain properties on binding to uPAR with inhibitory properties of enzymes relating to metastasis. However, experimental data relating to the metastasis inhibitory effect of this chimeric protein have not been reported.
Recently, the inventors found that human urinary tripsin inhibitor (UTI) inhibits invasion of cancer cells (27). UTI demonstrated not only invasion inhibitory effect of cancer cell in vitro (28), but also metastasis inhibitory effect in model system in vivo (29). In addition, the inventors found that xcex12-antiplasmin (xcex12-AP) and xcex12-macroglobulin (xcex12M), which are plasmin inhibitors belonging to a serpin family, do not inhibit a plasmin activity on plasma membrane, and that UTI inhibited a plasmin activity on plasma membrane leading to inhibition of invasion of cancer cell (29).
UTI comprises two Kunitz-type inhibitor domains and sugar chains (FIG. 2). A plasmin inhibitor site is located in HI-8, which is a second domain (residue 78-143 of UTI) on C-terminal side of UTI (30). The inventors demonstrates that HI-8 has a metastasis inhibitory activity (31). Recent research confirmed that HI-8 inhibited invasion and metastasis under mechanisms other than protease inhibitory action. HI-8 inhibits invasion of cancer cell, on the surface of which is not proved to have a plasmin activity. HI-8 is believed to inhibit invasion and metastasis of cancer cells by protease inhibitory action, and also inhibition of influx of calcium ion and regulation of protein kinase C (PKC) activity.
The inventors produced crosslinked compounds wherein ATF was chemically bound to UTI or HI-8 so as to improve an inhibitory effect by collecting UTI or HI-8 on cancer cells. The crosslinked compounds are found to inhibit metastasis of cancer cells in vitro effectively (32). The compounds synthesized by crosslinking agent, however, have a drawback in an industrial applicability that the compounds have crosslinks in a variety of manners leading to difficulty in large-scale production of substances with single structure.
An inhibitor of cancerous metastasis is a drug administered simultaneously in chemotherapy in case that primary tumor is removed by operation or that surgical treatment is difficult. In the cases, patients to be cured having decreased physical fitness can not tolerate drugs with potent toxicity. Recently, chemotherapeutic agents are revaluated in large scale from the viewpoint of decrease of self-healing ability due to side-effects of anti-cancer agents and of quality of life of patients during therapy.
UTI sample purified from human urine is used in medicinal application as curative medicine for acute circulatory failure and pancreatitis. UTI is a protein whose safety has already been confirmed in intravascular administration (33) (34) (35) (36). Since HI-8 is a part of UTI whose safety is confirmed, it is expected that HI-8 should be developed as cancerous metastasis inhibitor with low toxicity to human. In addition, G domain of uPA which is a region for binding to a receptor (uPAR) expressed in large amounts on metastatic cancer cells has actions of metastasis inhibition by inhibiting binding of uPA to cancer cells and of specific binding molecule to cancer cells. uPA is a substance which has already been developed as drug and has examined safety thereof. In view of foregoing, it is expected that a chimeric protein prepared by linking a polypeptide comprising G domain of uPA with HI-8 should have effective metastasis inhibitory actions based on combined properties of two proteins. In addition, the chimeric protein which utilizes partial sequences concerning specific functions of two drugs whose safety are established will be used as cancerous metastasis inhibitor with lower toxicity. Furthermore, a large scale production of the chimeric protein as substance having single structure of one polypeptide chain according to gene engineering techniques will greatly contribute to research of cancerous metastasis inhibition and development of inhibitor.